Taxonomic significance of epidermal and venation characters in the genus Diospyros L. (Ebenaceae) in Nigeria

This study investigated the foliar anatomy and venation patterns of thirteen species of the genus Diospyros in Nigeria with a view to identifying characters of great taxonomic value in the genus. Plant collections were made from different locations in Southwest Nigeria and the site of collection georeferenced with a GPS device. Samples were identified at the Herbarium of Botany Department, Obafemi Awolowo University (IFE). Voucher Specimens were deposited in the herbarium and voucher number assigned to each of the species. The samples were subjected to foliar epidermal and venation examinations. Qualitative data were recorded as the quantitative data were subjected to statistical analysis and the characters vary significantly (P = 0.05). Epidermal and venation characters which separate the taxa include stomata type, size and index, shape and size of epidermal cell, trichome type, presence/ absence of crystal/druses, as well as venation. Paxillate venation in D. suaveolens is diagnostic for the species. The presence of parenchymatous sheath over the veins and veinlets in D. tricolor is a spot character. The study reveals that leaf epidermal and venation characters are important in delimitation of species in the genus Diospyros.


Introduction
The genus Diospyros belongs to the family Ebenaceae (Ebony family). Ebenaceae is pantropical in distribution with about 500 species (Wallnofer, 2001). They are mainly represented by trees and shrubs with most species evergreen, but some are deciduous (Halle et al., 1978). In the family, leaves are simple, usually alternate, rarely opposite to sub opposite or in pseudowhorls of three, spirally or distichously arranged, petiolate or rarely sub sessile, induplicate or involutes in bud; stipules absent; lamina of leaves is coriaceous in many species, less frequently chartaceous, pinnately veined; leaf margins usually entire, very seldom finely crenulate, usually with strongly revolute margins at base, at least when dry; leaf apices spinose in some species.
The Ebenaceous inflorescence is conventionally referred to as a cyme (Ng, 1991). Solitary flowers are always terminal on the peduncle. Usually, male flowers of a given species by far outnumber the females. The actinomorphic flowers range from trimerous, pentamerous to octamerous. The number of floral parts is known to vary within species and cannot, therefore, be used effectively for their distinctions, as has been done in the past (Wallnofer, 2001). The fruit is 1-16 seeded multiloculate, usually indehiscent berries which is subtended by a persistent 3-8-lobed calyx. White (1983) posited that fruits of some African species of Diospyros sect. Royena become dry, and occasionally tardily dehiscent. In no species of Ebenaceae, however, is the fruit completely and spontaneously dehiscent. Seeds are often dispersed by various fruit-eating animals, such as civet cats, monkeys (Tutin et al., 1996). Ebenaceae are the source of several economically important products, the most valuable being their fruits and timber (ebony) and the genus Diospyros is best known for producing dense black wood (ebony) and tasty fruit (e. g. persimmons and zapotes) (Casper, 2013).
Most of the revisions of the genus Diospyros have been based on field observation and macromorphological characters (Wickremasinghe and Herat, 2006). In view of the complex taxonomic status in the genus, this study therefore set out to describe the leaf epidermal morphology and venation pattern of thirteen species of the genus Diospyros with a view to providing useful additional information for the delimitation and subsequent identification of members of the genus, thereby enhancing the taxonomic revision of the genus. The representative species of the genus include D. undabunda, D. mespiliformis, D. iturensis, D. canaliculata, D. precatorium, D. physocalycina, D. crassiflora, D. tricolor, D. suaveolens, D. dendo, D. conocarpa, D. nigerica and D. barteri.

Study area and target species
The present study was carried out in south-western Nigeria between Longitude 2° 31' and 6° 00' E and latitude 6° 21' and 8° 37' N (Agboola, 1979). The climate of South-Western Nigeria is majorly tropical, characterised by wet and dry seasons with temperature ranging between 21 °C and 34 °C and annual rainfall of about 150 mm to 3,000 mm (Faleyimu et al., 2013). Collection of plants was made from different locations in Southwest Nigeria and the site of collection was georeferenced with a GPS device (Table 1, Figure 1). Thirteen species were considered in this study, five accessions were considered for each species and ten matured leaves examined from different parts of the plants for each of the accessions.

Epidermal study
Sizeable portions from the leaves of the species studied were taken from standard median portion of the leaf while the scrape method of Metcalfe (1968) was used to obtain the epidermis. After scraping, the scraped portion was carefully cut and the peels were stained in Safranin O, rinsed in three to four changes of water and then mounted in 25% glycerol on a clean glass slide for light microscopy.

Venation pattern
Sizeable portions of the matured leaf of each of the species were obtained from the standard median portion of the leaf. The materials were decolorized by boiling in 90% ethanol at 20 °C for about 10-15 min (to remove chlorophyll), then washed in 3-4 changes of water to remove all traces of alcohol. The portions were then transferred to 5% Sodium Hydroxide and boiled for 30 min for further decolourization. The materials were later washed thoroughly to remove alkaline solution. The partially cleared leaves were further cleared in 5% domestic bleach (sodium hypochlorite) for 20-30 min under sunlight The portions were again washed in 3-4 changes of water, stained in Safranin O and counter stained in Alcian Blue (to enhance contrast), rinsed with water (to remove excess stain) before being mounted in 25% glycerol on a clean slide for examination under light microscope.

Foliar Epidermal Morphology and Venation Pattern
Leaves in the taxa studied are generally hypostomatic and it is considered a character of unification in the genus, hence their generic classification. However, while this finding contradicts the work of Ng (1971), who reported that stomata can be found on both surface of the leaf in D.  Figure 3B, E, F, K and L). Significant variations exist on both the type of stomata and distribution in the genus studied. The species can be grouped into four on the account of the type of stomata present. D. physocalycina and D. dendo are classified by anomocytic stomata on the abaxial surface ( Figure 3F and I). This observation is also in line with Schadel and Dickson (1979). D. canaliculata and D. conocarpa are grouped by staurocytic stomata ( Figure  3C and K), while D. mespiliformis and D. precatorium are characterized by paracytic stomata (Figure 3B and E), hence classificatory for the species. Other species as D. undabunda, D. iturensis, D. crassiflora, D. tricolor, D. suaveolens, D. nigerica and D. barteri are classified by cyclocytic stomata (Figure 3A, D, G, H, J, L and M). However, the presence of cyclocytic stomata in some species of Diospyros has previously been reported by Stace (1965) andWallnofer (2001) who both posited that some species in the genus Diospyros possess cyclocytic stomata and that their subsidiary cells differ somewhat in shape, size and orientation, and in having straight anticlinal walls. The quantitative foliar epidermal character of D. mespliformis shows a significant longer and wider stomata length and width as D. dendo possesses the least, D. precatorium also has the highest stomata density with D. crassiflora having the least hence separating the species (Table 2). The influence of trichome morphology and distribution in the taxonomy of different groups of plants has been well documented by Adedeji et al. (2007) in the delimitation of genera and species within the family Solanaceae. Rammaya and Rao (1976) and Rao and Rammaya (1977) have also emphasized the taxonomic importance of trichomes in the family Malvaceae. In this study, the presence of scales on the abaxial surfaces of D. dendo and D. conocarpa is classificatory for the two species ( Figure  3J and K). This reflects their level of closeness and monophyletic origin. The presence of scales in the two species aligns with the report of Wallnofer (2001) who reported that scale hairs are present on some African species of Ebenaceae.
Foliar venation has proven to be useful in delimitation of species (Levin 1986a). Leaf venation among many other characters of leaf is considerably different in each species, though constant among members of the same species (Provance and Sanders, 2006). In this study, irregular as well as welldeveloped triangular to polygonal areolation pattern which are peculiar and prominent in all the species studied suggests a generic character. The presence of 0 -6 vein density per areole in D. precatorium is a spot character for the species ( Figure 4E). The imperfect areole in D. suaveolens ( Figure 4I) is diagnostic for the species as all others possess perfect areoles. The species can be grouped into two on the basis of their nature of veins and veinlets: Simple or unbranched veinlets found in D. suaveolens and D. barteri is classificatory ( Figure 4I and M) while branched veinlets found in the rest of the taxa can be used in grouping them. D. dendo is distinctly separated from the rest of the species by the possession of significantly longer and wider areole (Table 3). On the contrary D. suaveolens is uniquely delineated from other species by the possession of significantly smaller areole size (Table 3). The occurrence of numerous druses in the areoles of D. suaveolens and D. barteri classifies the taxa and indicates a level of taxonomic relationship between the two species ( Figure 4I and M). Meanwhile, the importance of crystals in taxonomy has been highlighted by many scientists including Amos (1951) as well as Illoh and Inyang (1998). Ter Welle (1976) also emphasized that the mode of distribution of crystals is also an important taxonomic tool. The presence of parenchymatous sheath only in D. tricolor ( Figure 4H), as well as paxillate venation in D. suaveolens ( Figure 4I) (as other species are grouped by their regular polygonal reticulate venation) are diagnostic for the species and are of great taxonomic value. The dendogram of the species (Figure 5) revealed that D. mespiliformis and D. canaliculata as well as D. physocalicina and D. conocarpa are classified to the highest level of similarity as every other members of the genus (except D. undabunda) shared some generic relationship with them indicating they are recently evolved. However, D. undabunda is very distant to every other taxa hence its primitiveness. The similarity observed in D. mespiliformis and D. canaliculata as well as D. physocalicina and D. conocarpa is evident in their quantitative epidermal and venation characters and common ancestry. The PCA scattered diagram ( Figure 6) revealed the similarities in the genus. It clustered the species into two groups on the basis of their similarity and differences. The closeness observed between D. iturensis, D. nigerica, D. conocarpa, D. crassiflora and D. bateri and D. canaliculata as well as D. precatorium, D. canaliculata and D. mespiliformis is a function of the similarities in their leaf and venation taxonomic characters. This study therefore reveals that foliar epidermal and venation characters are important in delimitation of species in the genus Diospyros and should be used as basis for taxonomy.

Conclusion
This study concluded that foliar epidermis and venation characters are important in the taxonomy of the genus Diospyros. It has revealed additional information for the delimitation and subsequent identification of members of the genus, thereby enhancing the taxonomic revision of the genus.